LOCUS       sequence-431492-        2967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) guide expression; positive 
            control lethal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2967)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 2967)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA310
FEATURES             Location/Qualifiers
     source          1..2967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        444..692
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     444..464
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     615..634
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(877..893)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(877..893)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(890..912)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    901..917
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(925..931,932..949,950..955))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    970..991
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1108..1125)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1279..1867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1359..1378)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2045..2854)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2768..2787
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2846..2865
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagacgtc agagagggcc tatttcccat gattccttca tatttgcata
      481 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
      541 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      601 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      661 ctttatatat cttgtggaaa ggacgaaaca ccggttggag ctctgccgcc ggagtttaag
      721 agctatgctg gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa
      781 agtggcaccg agtcggtgct tttttgaaca cgcgtcttca tcggatcccg ggcccgtcga
      841 ctgcagaggc ctgcatgcaa gcttggtgta atcatggtca tagctgtttc ctgtgtgaaa
      901 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
      961 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     1021 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1081 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1141 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     1201 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     1261 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     1321 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     1381 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     1441 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1501 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1561 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1621 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1681 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     1741 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1801 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1861 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     1921 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1981 ttaaaaatga agttttaaat caagcccaat ctgaataatg ttacaaccaa ttaaccaatt
     2041 ctgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     2101 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     2161 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     2221 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     2281 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     2341 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     2401 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     2461 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     2521 gatattcttc taatacctgg aatgctgttt ttccggggat cgcagtggtg agtaaccatg
     2581 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     2641 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     2701 gaaacaactc tggcgcatcg ggcttcccat acaagcgata gattgtcgca cctgattgcc
     2761 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     2821 gcggcctcga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2881 aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga
     2941 gattttgaga cacgggccag agctgca
//
